Reengineering mRNA lipid nanoparticles for systemic delivery to pancreas

Abstract Lipid nanoparticles (LNPs) hold transformative potential for nucleic acid delivery, with applications ranging from clinical use, particularly in COVID-19 vaccines, to gene therapy and cancer immunotherapy. Traditional LNPs are composed of four components: ionizable lipids, cholesterol, helper lipids, and PEGylated lipids. However, a significant hurdle remains the need for more efficient and selective systemic delivery vehicles, as most targets are challenging to reach and primarily accumulate in the liver following intravenous administration, largely due to Apolipoprotein E (ApoE) mediated uptake in the blood. Recent studies have shown that introducing cationic or anionic lipids as a fifth component in the LNPs has resulted in lung- and spleen-specific mRNA expression. In this study, we report that incorporating endogenous ligands, such as vitamins, as a fifth component can enhance the extrahepatic localization of LNPs. Vitamins are highly biocompatible, possess excellent targeting potential, influence immune response, improve cellular uptake, and increase the stability of mRNA LNPs. We developed a library of 100 LNPs containing luciferase mRNA by partially replacing ionizable lipids with this fifth component and evaluated their efficacy both in vitro and in vivo. From comprehensive batch analysis screening, we identified two formulations with cholecalciferol (vitamin D3) as a fifth component that demonstrated selective systemic delivery to the pancreas (> 99%) with high efficacy. Among these, C-CholF3 emerged as the top formulation, exhibiting robust and sustained protein expression in the pancreas for up to 3 days in a dose-dependent manner with minimal toxicity that makes it suitable for repeated administration. Furthermore, C-CholF3 also demonstrated pancreas-specific gene editing in the Ai14 transgenic mouse model, showing high expression of TdTomato. This underscores its translational potential for protein replacement and CRISPR/Cas9-mediated gene editing in currently incurable pancreatic diseases.